Abstract

Introduction and Aims

Efficacy of targeted anticancer drugs is limited by de novo resistances related to cross talk within and between complex signal transduction networks. Rational combinatorial targeted therapies are one of the main solutions to increase activity or overcome resistance. We aimed to investigate the effects of inhibiting different nodes within the MAPK pathway (RAF and MEK) and PI3K-AKT pathway (PI3K, AKT and m-TORC1/2) either alone or in different combinations, on cell growth in a panel of KRAS mutant and KRAS wt NSCLC cell lines.

Material & Methods

We used dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 respectively. Inhibition of signalling output in MEK, PI3K, AKT and m-TORC1/2 was assessed by maximal reduction of p-ERK, p-AKT (Thr308), p-AKT (Ser473) and p-S6 by trametinib, GDC-0941, MK2206 and AZD2014 respectively. In keeping with its mechanism of action, maximal induction of p-MEK was considered as inhibition of RAF by dabrafenib in the cell line panel (none had mutations in BRAF). Quantification of phosphoproteins was done using MesoScale Discovery ELISA. Using concentrations of drugs shown to inhibiting signalling though these nodes, the effects of inhibiting the nodes alone or in combination on cell growth was studied using WST-1 assays. The NSCLC cell line panel included KRAS wild-type (H522; H1838; H1651) or KRAS mutant (A-549; Calu-6; H23) cells.

Results

The concentration range of dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 related to the different cell lines was [40-1000 nM], [40-200 nM], [1000 - 10 000 nM], >50 000 nM, and [500-1500 nM] respectively. The degree of growth inhibition following inhibition of an individual node varied between 5% and 75%. Highest inhibitions of cell proliferation by drugs alone were obtained with PI3K-AKT pathways compared to the MAPK pathway inhibitors for 6/6 cell lines. Drug combinations improved inhibition of cell proliferation in all cell lines. In 3/3 KRAS mutant cells, best combinations included MEK inhibitor and any other of the PI3K-AKT pathway as compared to inhibition of different nodes within the PI3K-AKT or MAPK pathways. In 3/3 KRAS wt cell lines, vertical combinations within the PI3K-AKT pathway induced highest inhibitions of cell proliferation compared to vertical combinations within the MAPK pathway or horizontal combinations between the PI3K-AKT and MAPK pathways.

Discussion/Conclusions

In the cell line panel tested, KRAS mutant cell lines were more sensitive to horizontal combinations than vertical combinations with the MAPK and PI3K-AKT pathways. KRAS wt cells were more sensitive to vertical combinations within the PI3K-AKT pathway. This preclinical study has clinical implications while planning combinations of targeted agents to treat NSCLC.